Journal: bioRxiv

Article Title: Oxysterol-liver X receptor signaling mediates CYFIP1 regulation of cortical neurogenesis

doi: 10.1101/2023.06.23.546272

Figure Lengend Snippet: A) Schematic representation of human CYFIP1 expression vector. B) qPCR quantification of CYFIP1 transcript levels in independent transgenic CYFIP1-GoF hESCs lines (TG#1 -10) and parental H7 cells (CTRL) at undifferentiated state. Data were normalised to the CTRL which was set as 1. C) qPCR analysis of CYFIP1 transcript during neuronal differentiation. Data shown are the mean between clones #3 and #5, all normalised to the CTRL cells at d5 of neuronal differentiation. D) Western blot confirming increased CYFIP1 protein level in CYFIP1-GoF cells (Clone #3) throughout 50 days of neuronal differentiation. Quantitative data on the right panel represent the average of clones #3 and #5. E) Schematic of CYFIP1 gene locus highlighting the first exon where gRNA sequences were designed. For space reasons, the sequence of the insertion present in one of the alleles of clone#70 is not reported and is indicated as “--200bp--”. F) DNA sequence flanking the edited site in the wild-type and edited CYFIP1 locus. G) Western blot demonstrating reduced CYFIP1 protein levels in 3 of the targeted clones (CYFIP1 clone#31, clone#41 and #70) compared to the parental line (CTRL).

Article Snippet: For CYFIP1 overexpression (CYFIP1-GoF), human CYFIP1 open reading frame (SC100426, OriGene) was cloned into a pCAG-IRES-PAC plasmid upstream of the IRES sequence [1].

Techniques: Expressing, Plasmid Preparation, Transgenic Assay, Clone Assay, Western Blot, Sequencing

Journal: bioRxiv

Article Title: Oxysterol-liver X receptor signaling mediates CYFIP1 regulation of cortical neurogenesis

doi: 10.1101/2023.06.23.546272

Figure Lengend Snippet: A) Outline of the cortical differentiation paradigm. B) Immunostaining for PAX6 (red) and NeuN (green) for CYFIP1-GoF, CYFIP1-LoF and their respective isogenic controls at d35 of differentiation. C-D) Quantification of PAX6 + and NeuN + cells shown in B. E-F) Day 20-50 cortical cultures were immunostained for cortical neuronal markers TBR1 (red), CTIP2 (green) and SATB2 (red), shown are representative images of day 30 staining. G-H) Quantification of TBR1 + , CTIP2 + and SATB2 + of day 20 to day 50 cortical cultures, respectively. Data presented are mean±s.e.m. from at least three biological replicates, groups were compared by Student t-test between the CYFIP1-manipulated vs isogenic control at each time point (*p<0.05; **p<0.01; ***p<0.001). Nuclei were counterstained with DAPI (blue). Scale bars: 50 µm.

Article Snippet: For CYFIP1 overexpression (CYFIP1-GoF), human CYFIP1 open reading frame (SC100426, OriGene) was cloned into a pCAG-IRES-PAC plasmid upstream of the IRES sequence [1].

Techniques: Immunostaining, Staining, Control

Journal: bioRxiv

Article Title: Oxysterol-liver X receptor signaling mediates CYFIP1 regulation of cortical neurogenesis

doi: 10.1101/2023.06.23.546272

Figure Lengend Snippet: A-B) Immunostaining for PAX6 (red) and FOXG1 (green) in d15 CYFIP1-GoF and CYFIP1-LoF NPCs cultures and respective controls. C-D) Quantification of PAX6 + and FOXG1 + cells. Data represent mean±s.e.m. from three biological replicates and analysed by Student t-test. No significant differences were found. Nuclei were counterstained with DAPI (blue). Scale bars: 50 µm.

Article Snippet: For CYFIP1 overexpression (CYFIP1-GoF), human CYFIP1 open reading frame (SC100426, OriGene) was cloned into a pCAG-IRES-PAC plasmid upstream of the IRES sequence [1].

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Oxysterol-liver X receptor signaling mediates CYFIP1 regulation of cortical neurogenesis

doi: 10.1101/2023.06.23.546272

Figure Lengend Snippet: Immunostaining and quantification for the proliferation marker Ki67 (green) and the cell cycle regulator p27 kip (red) in CYFIP1-LoF and isogenic iCas9 control progenitor populations. Nuclei were counterstained with DAPI (blue). Scale bars: 50 µm. Groups were compared by Student t-test (*p<0.05; **p<0.01).

Article Snippet: For CYFIP1 overexpression (CYFIP1-GoF), human CYFIP1 open reading frame (SC100426, OriGene) was cloned into a pCAG-IRES-PAC plasmid upstream of the IRES sequence [1].

Techniques: Immunostaining, Marker, Control

Journal: bioRxiv

Article Title: Oxysterol-liver X receptor signaling mediates CYFIP1 regulation of cortical neurogenesis

doi: 10.1101/2023.06.23.546272

Figure Lengend Snippet: A) Schematic illustration of the RNAseq experimental flow. B-C) Bar graphs showing examples of DEGs related to neural progenitors and neuronal differentiation at NPC2 (B) and neuronal stage (C), respectively. Bar height represents fold change (FC) the shade of color represents the FDR adjusted p value. D-E) Gene ontologies (GO) enriched in the DEG datasets associated with CYFIP1-GoF (D) and CYFIP1-LoF (E), compared to the respective control samples for all developmental stages analysed. Bar height represents the -log10 of the Benjamini-Hochberg adjusted P value, red line shows the threshold of significance (i.e. padj < 0.05). F) Bar graphs showing DEGs involved in cholesterol biosynthesis (green) and metabolism (red) and the master transcription factors for fatty acid and cholesterol biosynthesis (blue) at NPC2 and neuronal stages.

Article Snippet: For CYFIP1 overexpression (CYFIP1-GoF), human CYFIP1 open reading frame (SC100426, OriGene) was cloned into a pCAG-IRES-PAC plasmid upstream of the IRES sequence [1].

Techniques: Control

Journal: bioRxiv

Article Title: Oxysterol-liver X receptor signaling mediates CYFIP1 regulation of cortical neurogenesis

doi: 10.1101/2023.06.23.546272

Figure Lengend Snippet: A-B) PCA plot of CYFIP1-LoF, CYFIP1-GoF and their respective isogenic control cultures at d10 (NPC1), d20 (NPC2) and d40 (Neurons). C-D) Western blots analysis for CYFIP1, NIPA2, TUBGCP5 and GAPDH (internal control) on neural cells derived from control (CTRL) and 15q11.2-deleted iPSCs (EA8 and EA62), at the NPCs and neuronal stage. The quantitative protein level is expressed as intensity of the band corresponding to the protein of interest, normalised to the intensity of the GAPDH band on the same gel. Groups were compared by one-way ANOVA, followed by Tukey post-hoc test (*p<0.05). E) PCA for NPCs and neurons derived from 15q11.2del iPSC (EA8 and EA62) and two control iPSC lines. PC1 captures the largest difference segregating the different developmental stages while PC2 captures differences between genetic backgrounds. C) Bar graphs showing relative levels of 15q11.2 CNV transcripts to respective control cells at each of differentiation stage analysed. Bar height represents fold change (FC)

Article Snippet: For CYFIP1 overexpression (CYFIP1-GoF), human CYFIP1 open reading frame (SC100426, OriGene) was cloned into a pCAG-IRES-PAC plasmid upstream of the IRES sequence [1].

Techniques: Control, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Oxysterol-liver X receptor signaling mediates CYFIP1 regulation of cortical neurogenesis

doi: 10.1101/2023.06.23.546272

Figure Lengend Snippet: A) Bar graph showing the levels of 24S,25-EC produced by CYFIP1-GoF, CYFIP1-LoF, EA8 and EA62 15q11.2del iPSC and respective control PSC-derived neural cells at d20, 30 and 40. B) Schematic illustration showing 24S,25-EC treatment time windows. C) Representative images of immunostaining for TBR1, CTIP2 and NeuN at day 25 H7 cultures treated with ethanol vehicle control, and 24S,25-EC between day15-25. D) Quantification of the immunocytochemical analysis represented in C. E-G) Cumulative EdU incorporation assay of H7 cortical progenitor cultures treated with vehicle and increasing doses with 24S,25-EC from d15. Length of S-phase (F) and total cell cycle (G) determined by Nowakowski equation for all conditions. H) Quantification of TBR1 + , CTIP2 + and NeuN + in d25 CYFIP1-GoF cultures after 10 days exposure to increasing dose of 24S,25-EC or ethanol control. The red dashed line indicates the baseline (untreated) level in the isogenic control line. Data shown are mean±s.e.m. from three biological replicates per measurement (A) or treatment (D, F-H). Groups were compared by Student t-test between the indicated genotype and respective control per time point in A and one-way ANOVA followed by Tukey correction for D, F-H (*p<0.05; **p<0.01; ***p<0.001). Nuclei were counterstained with DAPI (blue). Scale bars: 50 µm.

Article Snippet: For CYFIP1 overexpression (CYFIP1-GoF), human CYFIP1 open reading frame (SC100426, OriGene) was cloned into a pCAG-IRES-PAC plasmid upstream of the IRES sequence [1].

Techniques: Produced, Control, Derivative Assay, Immunostaining

Journal: bioRxiv

Article Title: Oxysterol-liver X receptor signaling mediates CYFIP1 regulation of cortical neurogenesis

doi: 10.1101/2023.06.23.546272

Figure Lengend Snippet: A-B) Illustration of experimental scheme, immunocytochemistry and quantification for TBR1, CTIP2 and NeuN in d30 H7 cultures treated with 24S,25-EC from d20. C-D) Illustration of 24S,25-EC treatment scheme for iCas9 iPSC line, immunostaining and quantification for TBR1, CTIP2 and NeuN in d25 iCas9 cultures. E) immunocytochemistry for Ki67 4 days after 24S,25-EC or ethanol exposure. F) Number of Ki67 + and phosphorylated histone H3 + (pH3 + ) cells in H7 cultures treated with or without increasing dose of 24S,25-EC for 4 days from d15. G) Quantification of TBR1 + , CTIP2 + and NeuN + in d25 CYFIP1-GoF and H7 control cultures after 10 days exposure to increasing dose of 24S,25-EC or ethanol control. Data shown are mean±s.e.m. from three biological replicates. Shown in ‘G’ are the same data of presented as fold change of 24S,25-EC treated cultures to none treated cultures of CYFIP1-GoF (light blue) and isogenic H7 control (dark blue), respectively. Groups were compared by one-way ANOVA followed by Tukey correction (*p<0.05; **p<0.01; ***p<0.001). Nuclei were counterstained with DAPI (blue). Scale bars: 50 µm.

Article Snippet: For CYFIP1 overexpression (CYFIP1-GoF), human CYFIP1 open reading frame (SC100426, OriGene) was cloned into a pCAG-IRES-PAC plasmid upstream of the IRES sequence [1].

Techniques: Immunocytochemistry, Immunostaining, Control

Journal: bioRxiv

Article Title: Oxysterol-liver X receptor signaling mediates CYFIP1 regulation of cortical neurogenesis

doi: 10.1101/2023.06.23.546272

Figure Lengend Snippet: A) Bar graph showing the number of differentially expressed LXRβ and RXR target genes in CYFIP1-GoF, CYFIP1-LoF and 15q11.2del NPCs and neurons. LXRβ and RXR targets were compared to non-target DEGs by one-sided Fisher’s exact test (*p<0.05; **p<0.01; ***p<0.001). B-C) 24S,25-EC treatment during d15-25 failed to promote neuronal production of LXRβ-deficient NPCs. D-E) Premature neuronal differentiation of CYFIP1-LoF NPC is reversed by compound deletion of LXRβ and CYFIP1. Data shown in C and E are mean±s.e.m. from three biological replicates per treatment (C) or genotype (E). Groups were compared by one-way ANOVA followed by Tukey correction (*p<0.05; **p<0.01; ***p<0.001). Nuclei were counterstained with DAPI (blue). Scale bars: 50 µm.

Article Snippet: For CYFIP1 overexpression (CYFIP1-GoF), human CYFIP1 open reading frame (SC100426, OriGene) was cloned into a pCAG-IRES-PAC plasmid upstream of the IRES sequence [1].

Techniques:

Journal: bioRxiv

Article Title: Oxysterol-liver X receptor signaling mediates CYFIP1 regulation of cortical neurogenesis

doi: 10.1101/2023.06.23.546272

Figure Lengend Snippet: A) Schematic of LXRβ gene locus highlighting exon 4 where gRNA sequences were designed. B) Detection of InDels (insertions and deletions) via Sanger sequencing of the targeted exon 4 in iCas9 and CYFIP1-LoF background, respectively. C) Western blot analysis for LXRβ protein in iCas9 isogenic control, CYFIP1-LoF, LXRβ knockout and CYFIP1 & LXRβ double knockout iPSCs.

Article Snippet: For CYFIP1 overexpression (CYFIP1-GoF), human CYFIP1 open reading frame (SC100426, OriGene) was cloned into a pCAG-IRES-PAC plasmid upstream of the IRES sequence [1].

Techniques: Sequencing, Western Blot, Control, Knock-Out, Double Knockout